The dengue type 4 virus (DEN4) positive strand RNA genome contains a 384 nucleotide (nt) 3' nonc8oding sequence in which the last 81 nts, predicted to form a secondary structure, are thought to be essential for virus replication. Immediately upstream of the secondary structure short RNA sequences that are conserved among mosquito-borne flaviviruses have been identified. A series of deletions that range from 30 to 262 nts was introduced into this region of full-length DEN4 cDNA to create viable deletion mutants some of which might prove to be useful for inclusion in a live attenuated virus vaccine. When studied by an infectious center assay most full length RNA transcripts of the deletion constructs exhibited reduced infectivity when transfected into simian LLC-MK2 cells compared to the full-length RNA transcripts of wild type parental virus. Deletion mutations that extended as far as the 5' boundary of the 3' noncoding region and whose 3' boundary did not extend beyond the last 113 nts of the 3' end were viable. With the exception of mutant 3'd 303-183 which contained a deletion of the region that was 303 to 183 nts from the 3' terminus, deletion mutants produced plaques that appeared late on simian LLC-MK2 cells or exhibited a small plaque morphology on mosquito C6/36 cells, compared to the wild type parental virus. These mutants also replicated less efficiently and attained a lower titer in LLC-MK2 cells than parental wild type virus. Significantly, mutant 3'd 303-183 grew to a high titer and was least restricted in growth. Mutant 3'd 303-183 and four other moderately to severely restricted mutants were selected for evaluation of infectivity and immunogenicity in rhesus monkeys. There was a suggestion that occurrence and duration of viremia was reduced for some of the deletion mutants compared to the wild type virus. However, more convincing evidence for attenuation of some of the mutants was provided by an analysis of antibody response to infection. Mutant 3'd 303-183 induced an antibody response equivalent to that stimulated by wild type virus, whereas other mutants induced low to moderate levels of antibodies as measured by radioimmuno-precipitation and virus neutralization. The immunogenicity of these 3' DEN4 deletion mutants in monkeys appeared to correlate with their efficiency of growth in simian LLC-MK2 cells. One or more mutants developed during this project may prove to be useful for immunization of humans against disease caused by dengue virus.